\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{16a-Bromoepiandrosterone as a new candidate for diabetes-tuberculosis
comorbidity treatment}



\author[1]{Manuel Othoniel Lopez Torres}%
\author[2]{Brenda Marquina}%
\author[3]{Octavio Ramos-Espinosa}%
\author[1]{Dulce Mata}%
\author[4]{Jorge Barrios-Payan}%
\author[5]{Guillermina Baay}%
\author[6]{Sara Huerta-Yépez}%
\author[7]{Cristian Alfredo Segura-Cerda}%
\author[8]{Estela Bini}%
\author[1]{Ivan Torre-Villalvazo}%
\author[1]{Nimbe Torres}%
\author[1]{Armando R Tovar}%
\author[9]{William  Chamberlin}%
\author[9]{Yu Ge}%
\author[10]{Andrea  Carranza}%
\author[2]{Rogelio Hernandez Pando}%
\affil[1]{Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran}%
\affil[2]{National Institute of Medical Sciences and Nutrition}%
\affil[3]{National Institute of Medical Sciences and Nutrition Salvador Zubirán}%
\affil[4]{National Institute of Medical Sciences and Nutrition “Salvador Zubiran”}%
\affil[5]{Oncological Diseases Research Unit, Hospital Infantil de Mexico "Federico Gomez", Mexico City, Mexico.}%
\affil[6]{Hospital Infantil de Mexico "Federico Gomez"}%
\affil[7]{Centro de Investigacion y Asistencia en Tecnologia y Diseno del Estado de Jalisco}%
\affil[8]{National Institute of Medical Sciences and Nutrition ‘‘Salvador Zubirán’’}%
\affil[9]{Protibea Therapeutics }%
\affil[10]{Universidad de Buenos Aires}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Being the leading cause of mortality by a single infectious agent,
tuberculosis (TB) continues as one of the most relevant issues of public
health. Another pandemic disease is type 2 diabetes mellitus (T2D) is
usually associated with immunodeficiency. Thus, an increased prevalence
of TB-T2D comorbidity represents one of the most significant challenges
for health providers. During the chronic phase of both diseases, several
immunoendocrine abnormalities are occurring, but extra-adrenal
production of active glucocorticoids (GCs) is a possible deleterious
factor in both entities. Active GCs have been related to insulin
resistance and suppression of Th1 responses, contributing to T2D and TB
pathogenesis. 11-\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase type 1 (11-\selectlanguage{greek}β\selectlanguage{english}HSD1)
catalyzes the conversion of inactive GGs in their active form (cortisol
or corticosterone in rodents) in the lungs and liver and could be
responsible for this immunoendocrine disfunction. Dehydroepiandrosterone
(DHEA) is an anabolic adrenal hormone with antagonist effects against
GCs on immune cells and glucose metabolism. A synthetic analog of DHEA,
the 16a-bromoepindrosterone (BEA), lacks an anabolic effect while
keeping his immune and metabolic effect. The therapeutic efficiency of
BEA was studied in a murine model of T2D-TB comorbidity. TB-T2D mice
underwent more severe lung disease than in TB-infected but non-diabetic
animals. BEA decreased the active form of GCs and 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 expression,
while increasing 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 expression, which reduced hyperglycemia and
liver steatosis, lung bacillary loads, and pneumonia. Thus, it seems
that BEA is an efficient therapy to control metabolic and immune
abnormalities caused by high active GCs production.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Type 2 Diabetes (T2D) is currently the most widespread susceptibility
factor for tuberculosis (TB) with a high incidence and prevalence in
low- and middle-income countries (1). In most cases, Mycobacterium
tuberculosis (Mtb) infection is controlled by immune mechanisms that
contribute to granuloma development and control of bacterial growth (2).
However, recent research on TB-T2D comorbidity shows that innate and
adaptive immunity are more affected when both conditions occur together
than when only one disorder is present. (3).

A type 1 T helper (Th1) cell response mediates the protective immune
response to TB through macrophages activation by IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF\selectlanguage{greek}α \selectlanguage{english}(4).
Observations in T2D patients show an impaired response to Mtb infection
(5), consisting in alterations in bacterial recognition, phagocytosis,
and a deficient immune cellular response with low cytokine and chemokine
production (6), resulting in severe TB manifestations (7).

A new aspect in understanding the higher susceptibility of T2D to
develop TB is the study of the abnormal metabolic factors and
neuro-immune-endocrine regulation in this co-morbidity. In homeostatic
conditions and under acute or chronic stress, the principal source of
glucocorticoids (GCs) are the adrenal glands regulated by the activation
of the Hypothalamus-Pituitary-Adrenal (HPA) axis (8). Besides this
source of circulating GCs, there is a local organ conversion of inactive
to active GCs (9), which is mediated by the enzyme 11-\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid
dehydrogenase type 1 (11-\selectlanguage{greek}β\selectlanguage{english}HSD1) that has an oxo-reductase activity using
NADP(H) as a cofactor. This enzyme converts inactive cortisone to active
cortisol in humans or corticosterone in rodents (10). Interestingly,
there is another enzyme with the opposite effect. 11-\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid
dehydrogenase type 2 (11-\selectlanguage{greek}β\selectlanguage{english}HSD2) acts exclusively as an NAD-dependent
dehydrogenase that converts active cortisol or active corticosterone to
inactive cortisone (11).

Although blood levels of glucocorticoids are in the normal range in T2D
and TB, it seems that this is not the case in the lung and liver. Higher
activity of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 in these peripheral organs results in increased GC
production in these organs.

GCs are critical homeostatic hormones that are overproduced after
diverse environmental insults, and in general they have a negative
effect on metabolism and the immune response (12). GCs exert their
effects by binding to glucocorticoid receptors (GR) present in all cell
types. Hence, GR signaling plays an essential role in the modulation of
many biological functions in immune cells and several tissues, including
liver, lung, adipose tissue, thymus and muscle (13).

Cortisol suppresses the expression of pro-inflammatory and adhesion
molecules, thus preventing the extravasation of neutrophils to the site
of inflammation (13). Chronic exposure induces an anti-inflammatory gene
expression profile of resident macrophages and decreases their
phagocytic activity. Furthermore, active GCs suppress Th1 cytokine
production and induce cell death (14), promoting a Th2 environment that
impairs granuloma formation (7). GCs inhibit the proliferation of
effector T cells and induce apoptosis of neutrophils, basophils, and
eosinophils resulting in reduced inflammation (15). GCs also have an
important contribution in the physiopathology of TB. It has been
reported in an experimental model of progressive TB that at the time of
maximal protective activity mediated by IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}and NO production,
the hypothalamic-pituitary-adrenal axis is activated, thereby
stimulating the adrenal glands to secrete GCs (corticosterone) with the
apparent aim of avoiding tissue damage produced by excessive lung
inflammation. However, the excess of corticosterone also inhibits Th1
lymphocytes activity and induces differentiation of Th-2 lymphocytes.
This favors bacterial survival and proliferation, causing animals death
(14). As far as we know, there are no published studies about the
pulmonary production of active GCs mediated by 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 in TB.

In metabolic diseases such as obesity and metabolic syndrome, GCs play a
role in the development of diabetes, but interestingly, GCs in the
circulation have the same concentrations as healthy subjects (16).
Experimental studies with transgenic mice overexpressing 11-\selectlanguage{greek}β\selectlanguage{english}HSD1
selectively in adipose tissue demonstrate increased local levels of
corticosterone that induce visceral obesity development, insulin
resistance, diabetes, and hyperlipidemia (16). The increased conversion
of active glucocorticoids in visceral adipose tissue increases lipolysis
and fatty acid levels (17).

Moreover, active GCs are potent inducers of glycogenolysis and
gluconeogenesis, resulting in a hyperglycemic state, deteriorating the
condition of diabetic patients (18). 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 also increases glucose
output by activating phosphoenolpyruvate kinase (PEPCK) gene expression
in the liver (19). Thus, active GCs have direct implications in the
abnormal metabolic control and chronic complications of T2D (20).

Regulation of cortisol is mediated by dehydroepiandrosterone (DHEA), an
anabolic hormone of the adrenal cortex involved in the conversion of
sexual steroids. This hormone displays antagonistic activity to
cortisol, impacting the immune response (21) and glucose metabolism. The
cortisol-DHEA ratio is modified in T2D-TB comorbidity and represents a
deleterious factor for both diseases (8). 16\selectlanguage{greek}α-\selectlanguage{english}Bromoepiandrosterone (BEA)
is a synthetic analog hormone of DHEA that modulates immune and
metabolic responses (22, 23). Compared to DHEA, BEA does not display
anabolic activities.

Here, we studied the expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and 11-\selectlanguage{greek}β\selectlanguage{english}HS2, as well as the
concentrations of GCs during experimental late TB and T2D/TB in the lung
and liver and evaluate the therapeutic effect of BEA administration in a
murine model of T2D-TB comorbidity.

\textbf{Materials and Methods}

\emph{Ethics Statements}

All the animal work was done according to the guidelines of the Mexican
law NOM 061-Z00-1999 and approved by the Internal Committee for the Care
and Use of Laboratory Animals (CICUAL) of the National Institute of
Medical Sciences and Nutrition in M\selectlanguage{ngerman}éxico.

\emph{Experimental Model of Type 2 Diabetes and Progressive Pulmonary TB
in BALB/c Mice}

To induce T2D, a non-genetic mouse model of non-insulin-dependent
diabetes mellitus previously reported was reproduced (26). Briefly,
young male BALB/c mice three weeks old were fed with a high fat diet
(HFD) at 45\% of calories during the whole course of the experiment. At
3 months old, animals received a single low dose (100 mg/kg) of
estreptozotocin (STZ) ChemCruz (Santa Cruz Biotechnology, Dallas, TX) by
intraperitoneal (i.p) injection. That STZ dose did not cause diabetes in
chow-fed mice (26). We assessed the combined effect of HFD and STZ in
glucose blood levels with Accu-chek active (Roche diabetes care
Mannheim, Germany) test strips, after and before of STZ administration.
Serum cholesterol and triglyceride concentrations were measured with
Acu-trend one month after STZ administration.

After one month of STZ administration, animals showed hyperinsulinemia
and hyperglycemia that reverted by the treatment with oral hypoglycemic
drug (metformin), a similar metabolic pattern and respond to treatment
of T2D (Fig. 1). Animals with these metabolic abnormalities compatible
with T2D were infected by the intra-tracheal route with a high dose of
Mtb reference strain H37Rv (ATCC No. 25618), in order to induce
progressive pulmonary TB. Briefly, Mtb strain H37Rv was grown in
Middlebrook 7H9 broth (DIFCO) supplemented with 0.2\% glycerol, 10\%
OADC enrichment, and 0.02\% Tween-80 and maintained at 37\selectlanguage{english}*C in
agitation. Mid log-phase cultures were used for infection. Mycobacteria
were counted and stored at \selectlanguage{english}-80\selectlanguage{english}*C until use. Bacterial aliquots were
thawed and pulse-sonicated to remove clumps. For the infection, groups
of T2D and control animals fed with chow diet were anesthetized in gas
chamber using Sevofluorane and infected intra-tracheally with 2.5
A\textasciitilde{} 10\textsuperscript{5} live bacilli using a cannula
inside a biosafety level III cabinet. Mice were maintained in vertical
position until spontaneous recovery and maintained in groups of five in
cages fitted with micro-isolators connected to negative pressure in
animal biosafety level III facilities.

In order to compare the evolution of TB in T2D and non-diabetic mice,
groups of 5 mice were euthanized by exsanguination under pentobarbital
anesthesia after 14, 21, 28 and 120 days after infection. Lungs were
rapidly collected and immediately frozen by immersion in liquid nitrogen
and used for bacillary loads determination by colony forming units
quantification and TNF\selectlanguage{greek}α \selectlanguage{english}expression determined by RT-PCR, as described
below. Ten animals per group were left untouched and their survival was
recorded to construct survival curves.

\selectlanguage{greek}\emph{Expression and cellular source of 11-βHSD1, 11-βHSD2 and
glucocorticoids in the lungs and liver during late active pulmonary TB}\selectlanguage{english}

Groups of five mice with T2D, TB and T2D/TB were euthanized by
exsanguination under pentobarbital anesthesia after 60 and 120 days of
infection, in two independent experiments. The late active disease is
well established by this time in this model, as characterized by
extensive pneumonia, high bacillary loads and low expression of IFN-\selectlanguage{greek}γ
\selectlanguage{english}and TNF-\selectlanguage{greek}α. \selectlanguage{english}Three right lungs were intra-tracheally perfused with
absolute ethanol and after 24hr the tissue was embedded for histology
and immunohistochemistry studies, while the left lungs were immediately
frozen for total RNA isolation and gene expression determination of
11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 by RT-PCR. The same tissue samples for the
histology study were used to determine by immunohistochemistry the
cellular source of corticosterone and cortisone and their concentrations
by digital pathology. Tissue fragments from the liver were processed in
the same way.

\emph{BEA treatment in infected animals with and without T2D}

After two months of infection, treatment with BEA was started in groups
of mice with TB or T2D/TB administrated each other day .02 mg/kg by
intra-tracheal route to produce a direct effect on the infected lung,
and the same amount of BEA administrated by subcutaneous route for
systemic distribution promoting hepatic effect. TB group received BEA
only by intratracheal instillation. After 30 and 60 days of treatment,
groups of six animals were euthanized by exsanguination under anesthesia
with 210 mg/kg of intraperitoneal pentobarbital, inside in a cabinet of
biosecurity level III. Three left lungs per time were perfused with
absolute ethanol, fixed, and prepared for histopathological studies.
After eliminating hilar lymph nodes and thymic tissues, five lungs were
frozen and kept to -80*C for bacilli load quantification and three lungs
were used to determine the expression of diverse genes by RT-PCR. Two
independent experiments were performed. Animals were monitored daily and
humanely euthanized if exhibited respiratory insufficiency or
substantial weight loss.

\emph{Determination of colony-forming units in lung}

Right lungs from four mice at each time-point, in two different
experiments, were used for bacillary load determination by colony
forming counting (CFU) quantification. Lungs were homogenized with (MP
Biomedicals FastPrep-24) in 1mL of 0.05\% Tween-80 and the same
methodology was use with spleens after weighted them. Four consecutive
logarithmic dilutions were made from this homogenate. Ten microliters of
each dilution were plated in duplicate on Bacto Middlebrook 7H10 agar
(Difco, Detroit, MI, USA) enriched with OADC. Plates were incubated at
37\selectlanguage{ngerman}ºC and 5\% CO2 during 21 days for CFU quantification.

\emph{Preparation of lung tissue for histological and
immunohistochemistry analysis}

Lungs of mice were perfused with absolute ethanol by endotracheal route
and fixed for 24 h and then embedded in paraffin blocks. Sections of 4\selectlanguage{greek}μ\selectlanguage{english}m
width were obtained, mounted on glass slides, deparaffinized and stained
with hematoxylin and eosin. For quantification and morphometric analysis
determining the surface area affected by pneumonia, three different
mouse lungs per time point were evaluated in two independent
experiments.

For immunohistochemistry, lung tissue was sectioned and mounted on
charged glass slides and then deparaffinized. Slides were first blocked
for unspecific activity of peroxidase with methanol peroxide 3\% for 1
h. For 11-\selectlanguage{greek}β\selectlanguage{english}HSD1and 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 tissue detection, rabbit anti-mouse
monoclonal antibodies (Biorbyt) were used at a concentration of 1:250,
incubated overnight in agitation, followed by incubation with secondary
anti-rabbit IgG labeled with peroxidase. For cortisone and
corticosterone immunostaining, rabbit antimouse monoclonal antibodies
(LsBio) were used at a final concentration of 1:250. In both cases,
bound antibodies were detected with diaminobenzidine and counterstained
with hematoxylin.

Immunohistochemistry stained sections were analyzed by digital
pathology. Briefly, stained sections were digitized at 200 x
magnification using an Aperio ScanScope CS (Aperio). The Aperio
ScanScope CS images had a spatial resolution of 0.45 \selectlanguage{greek}μ\selectlanguage{english}m/pixels. The
images were analyzed using an ImageScope (Aperio). All the lung tissue
sections were circumscribed and sent for automated image analysis using
the Spectrum Software V11.1.2.752 (Aperio). For immunostaining GCs
intensity quantification, an algorithm was developed to determine the
total lung expression of cortisone and corticosterone in the whole
tissue section (26--28).

\selectlanguage{greek}\emph{Gene expression of 11-βHSD1, 11-βHSD2 and cytokines determined by
RT-PCR in lung and liver}\selectlanguage{english}

The right lungs of three mice per time point were rapidly obtained and
stored in 1.5-ml cryotubes, immediately frozen in liquid nitrogen and
maintained at -80*C until processing. For homogenization, lungs were
slowly defrosted, and zirconium flint beads were added to each tube and
lung tissue was homogenized in the FastPrepR-24 (MP Biomedicals). RNA
extraction was performed with the RNeasyR Mini kit (Qiagen) following
the manufacturer's instructions.

Total RNA was quantified by spectrophotometry (A260/280), 100 ng of RNA
from each lung was used for the production of cDNA by
retro-transcription, following the indications of the Omniscript kit
(Qiagen), and later an endpoint PCR was run to amplify the constitutive
housekeeping gene RPLP0 (Ribosomal protein large P0, Gen ID:11837,
GenBank, NCBI) and its integrity was analyzed by running at 2\% agarose
gel stained with SYBR green.

Complementary DNA (cDNA) obtained from each sample was analyzed by
real-time PCR (qPCR) using the Real-Time PCR system 7500 (Applied
Biosystems) and the Quantitech SYBR Green Mastermix kit (Qiagen) with
specific primers designed with the first BLAST (ncbi.nlm.nih.gov) for
11-\selectlanguage{greek}β\selectlanguage{english}HSD1, 11-\selectlanguage{greek}β\selectlanguage{english}HSD2, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α.

\selectlanguage{english}Regarding to relative quantification of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1, 11-\selectlanguage{greek}β\selectlanguage{english}HSD2, the Ct
values were determined by 7500 real-time PCR system (Applied Biosystems,
Foster City, CA, USA), and the fold change of gene expression was
calculated by the 2-(^^Ct) method (29). For absolute quantification, the
number of copies of each target gene was normalized to 1 million
amplicons of mRNA of the housekeeping gene RPLP0, including the standard
curves and a negative control to IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}and 11-\selectlanguage{greek}β\selectlanguage{english}HSD1, 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 in
liver. Cycling conditions used were as follows: initial denaturation at
95*C for 15min, followed by 40 cycles at 95*C for 20 s, 60*C for 20 s,
and 72*C for 34 s.

\textbf{Statistical Analysis}

All the statistical analysis was performed using GraphPad Prism Software
(version 6.0, La Jolla, USA). The data were analyzed using paired
t-test, one- and two-tailed ANOVA with Bonferroni correction for
multiple comparisons. P-values \textless{} 0.05 were considered
significant.

\textbf{Results}

\textbf{Comparative pulmonary disease evolution of TB \& T2D/TB}

Mice fed with hypercaloric diet for three weeks old and treated at eight
weeks old with only one low dose of streptozotocin showed progressive
weight increase (Fig. 1B), and after two months showed significant
higher sera concentration of glucose, triglycerides, cholesterol and
insulin (Fig. 1C, D, E, and F). The histology study of the liver showed
extensive lipid cytoplasmic vacuolization of hepatocytes (steatosis)
(Fig. 1H). Treatment of these mice with metformin or glibenclamide
resulted in normal blood concentrations of glucose (Fig. 1G). Thus,
these mice showed the metabolic parameters of T2D.

In comparison with chow fed mice infected with Mtb, infected T2D mice
showed significant higher pulmonary bacilli burdens (Fig. 2A). T2D/TB
mice also showed lower expression of TNF-\selectlanguage{greek}α \selectlanguage{english}and decreasing survival rate
than TB mice (Fig. 2B, and 2C). Thus, T2D/TB mice suffered more severe
disease than TB mice.

\selectlanguage{ngerman}\textbf{Comparative expression of GC´s and GC´s converting enzymes in
the late phase of TB \& T2D-TB lungs}

Before evaluating the therapeutic effect of BEA, comparison among
non-infected control mice fed with chow diet, T2D, TB and T2D-TB groups
was done in the lungs of animals with late disease at day 120 of
infection as determined by immunohistochemistry, the presence of
corticosterone (active GC) and cortisone (inactive GC), as well as the
genetic expression of the GC converting enzymes determined by RT-PCR and
their cellular source by immunohistochemistry (Fig. 3). The lungs of
control non-infected mice showed occasional bronchial epithelial cells
with corticosterone positive immunostaining, while in T2D mice a clear
increase in the number and intensity of corticosterone was seen in
airways epithelium (Fig. 3 A). In the TB and T2D-TB groups the bronchial
epithelium was negative, and the pneumonic areas showed intense
corticosterone immunostaining in some lymphocytes and numerous
macrophages, being many of them vacuolated or foamy macrophages (Fig.
3A). In comparison with the control non-infected group, there was also a
significant increase in the genetic expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 in T2D, TB
and T2D-TB groups, being macrophages from pneumonic areas the cells that
showed strong immunostaining to this enzyme (Fig. 3B). Thus, there is a
high expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and corticosterone production in the
inflammatory cells of pulmonary TB, which is even higher in mice with
T2D-TB.

Regarding inactive GC cortisone, the lungs from non-infected mice showed
positive immunostaining in the alveolar epithelium, which was lower in
T2D animals and quite strong in the TB lungs, while in T2D-TB was
slightly positive, almost negative (Fig. 3C). These immunohistochemical
results are well correlated with the 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 gene expression, which
showed significant higher expression in non-infected lungs, while it was
lower in TB, and the T2D-TB group showed the lowest (Fig. 3D).

\textbf{Effect of BEA in lung and spleen bacillary burdens, pulmonary
cytokine expression and tissue damage in TB and T2D-TB mice}

To evaluate the therapeutic effect of BEA, the TB and T2D-TB groups and
their respective control groups that received only the vehicle were
compared through bacillary loads in the lung and spleen (CFUs), extent
of tissue damage (percentage of lung surface affected by pneumonia), and
the gene expression of the protective cytokines TNF-\selectlanguage{greek}α \selectlanguage{english}and IFN-\selectlanguage{greek}γ
\selectlanguage{english}(RT-PCR). In comparison with their respective controls non-treated mice,
both T2D-TB and TB groups after one and two months of treatment with BEA
showed significant reduction of pulmonary bacillary burdens (Fig. 4A).
BEA induced an increase in gene expression of IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}after one
month of treatment in both groups, and IFN-\selectlanguage{greek}γ \selectlanguage{english}in TB group after two
months of treatment (Fig. 4B, and 4C); as well as reduction of
pneumonia. Interestingly, BEA treatment for two months induced in the
spleen significant reduction of CFUs and weight increase only in the
T2D-TB group (Fig. 4E, and 4F), which suggest spleen hyperplasia with
better control of bacillary dissemination in this experimental group.

\selectlanguage{greek}\textbf{Effect of BEA on the expression of 11-βHSD1 and 11-βHSD2,
corticosterone and cortisone in the lungs and liver.}\selectlanguage{english}

In the lung, treatment with BEA in TB and T2D-TB groups induced
significant lower expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 after one and two months of
treatment when compared with the control group (Fig. 5). This effect was
even more evident in the liver of T2D-TB group, which also showed
complete disappearance of steatosis and hyperglycemia (Fig. 6D).

The effects of BEA on 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 gene expression in the infected lung were
in concordance with the corticosterone immunehistochemistry staining,
and its quantitative evaluation by digital pathology (Fig. 5A). BEA
significantly decreased active corticosterone in TB and TB/T2D mice,
being more pronounced in the TB group (Fig. 5C). In contrast, the
expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 after one and two months of BEA treatment showed
a significant increase in the TB group, and cortisone was increased in
both groups (Fig. 5D, and 5E).

Thus, it seems that BEA is a regulator of the genetic expression of
these enzymes in the lung and liver, inducing lower expression of
11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and higher gene expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD2. In consequence BEA
induced lower production of active corticosterone and higher production
of inactive cortisone, which could contribute to increase the expression
of the pro-inflammatory cytokines TNF-\selectlanguage{greek}α \selectlanguage{english}and IFN-\selectlanguage{greek}γ \selectlanguage{english}decreasing the
pulmonary bacillary loads. In the liver BEA improved lipid and glucose
metabolism and normalized liver histology and glucose blood
concentrations.

Thus, BEA improved the immune response and metabolism of the comorbidity
T2D/TB, suggesting that it could be a potential new immune therapeutic
agent with beneficial immune, endocrine and metabolic activities.

\textbf{Discussion}

TB-T2D comorbidity is a new area of interest due to its recent increased
incidence worldwide. T2D is a metabolic endocrine disease associated
with abnormal secretion and action of insulin and chronic hyperglycemia
affecting multiple organ systems. T2D compromises host immune responses
and impairs host ability to control microbial infections. Indeed, T2D
produced several abnormalities in diverse immune mechanisms against Mtb
that worsens the course of the disease requiring longer courses of
chemotherapy (7, 8). Our results confirm the more aggressive course of
TB in an experimental model of T2D/TB; and for the first time
demonstrates that one significant pathogenic factor is the higher
expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and corticosterone production during late
advanced disease in the lungs and liver. The treatment with BEA
efficiently decreases the expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and antagonizes
corticosterone producing significant reductions of bacillary burdens and
hyperglycemia. These results suggest that this synthetic hormone can
contribute to the treatment of this co-morbidity.

Mtb affects mainly the lungs producing chronic and excessive
inflammation in which innate and adaptive immunity are deeply affected
(2, 3). Infection starts by inhalation of saliva droplets with Mtb that
are engulfed by alveolar macrophages, which are key cells in bacilli
elimination and together with dendritic cells process Mtb antigens that
are presented to T lymphocytes in regional lymph nodes. Lymphocytes
migrate to the lung and with macrophages and other cells form
containment structures known as granulomas which are the
histopathological hallmark of TB (5). In early stages of active
infection, Th1 cellular immune responses are protective, as IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF\selectlanguage{greek}α
\selectlanguage{english}and other cytokines such as IL-12 induce macrophage activation, allowing
bacterial growth control. Nevertheless, during late active disease,
extensive inflammation produces a shift toward anti-inflammatory
responses, such as Th-2 cytokine pattern in which IL-4, IL-13 and other
anti-inflammatory cytokines such as IL-10 and transforming growth
factor-\selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}) induce a local immunosuppressive/anti-inflammatory
milieu, resulting in poor containment of infection and progression of
tissue damage. Mortality rates approximate 1\%. (30). Interestingly,
besides these immunologic responses, there is an intense neuroendocrine
response that creates a complex network of cytokines, hormones, and
neurotransmitters that influence the outcome of TB pathogenesis (8, 9,
31). These neuroendocrine responses include abnormal production of
several hormones, that is partly mediated by the cytokines released
during the immune response to Mtb (pro-inflammatory mediators), and also
contribute to modify the central nervous system (CNS) response by
activating the two major stress systems, the HPA axis (32) and the
sympathetic nervous system (SNS) (7,33).

HPA and GCs have an important contribution to the physiopathology of TB
(34, 35). It was reported in the experimental model of progressive TB in
BALB/c mice that at the time of maximal protective activity mediated by
IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β \selectlanguage{english}and NO production (day 21 after infection),
pro-inflammatory cytokines such as TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}strongly activate the
HPA axis, producing high expression of CRF in the hypothalamus and
adrenal hyperplasia with high serum concentrations of corticosterone
(32). Then, during the chronic or late phase after 28 days of infection,
there is progressive adrenal atrophy and a corticosterone decrease in
sera co-existence with extensive pneumonia (32). Thus, there is high GCs
adrenal production apparently with the aim to avoid tissue damage
produced by excessive lung inflammation. During the late phase of the
disease when adrenal glands are atrophic, it seems that there is another
source of corticosterone available by production in peripheral
macrophages.

Our results show, for the first time, that this extra-adrenal source of
GCs are macrophages, particularly foamy cells which strongly express the
enzyme 11-\selectlanguage{greek}β\selectlanguage{english}HSD type 1 and serve as a new source of corticosterone in the
inflamed tuberculous lung. This local source of corticosteroids acts in
an autocrine and paracrine manner. However, this local excess of
corticosterone also inhibits Th1 lymphocyte activity and induces
differentiation of Th-2 lymphocytes that favors bacterial survival and
proliferation causing animals death (14). Thus, this local
anti-inflammatory activity mediated by high production of corticosterone
in affected organs during active late TB have deleterious effect.
Antagonistic hormones such as DHEA can restore balance to the system.

DHEA is the most abundant product of the human adrenal gland after
puberty with maximum levels occurring in the late 20s. Its production
then falls steadily with increasing age. DHEA has anti-glucocorticoid
properties in several systems. For example, DHEA shows opposite effects
to GCs on enzyme expression in the liver in obesity (1) and in the
immune system (4). In a previous study, treatment with DHEA starting on
day 60 after Mtb infection in BALB/c mice reduced the pulmonary
bacillary burden and decreased tissue damage by reactivating the Th-1
cytokine response (36). In humans a deficit in DHEA relative to cortisol
can be striking in severe human TB (8). However, DHEA is suboptimal for
human use, partly because it is metabolized into sex steroids.

16\selectlanguage{greek}α-\selectlanguage{english}Bromoepiandrosterone (BEA; 16\selectlanguage{greek}α-\selectlanguage{english}bromo-5\selectlanguage{greek}α-\selectlanguage{english}androstan-3\selectlanguage{greek}β-\selectlanguage{english}ol-17-one) is a
synthetic adrenal steroid derivative that does not enter sex steroid
pathways and when tuberculous BALB/c mice were treated with BEA, every
other day beginning on day 60, it produced a significant inhibition of
bacterial proliferation and higher expression of protective cytokines
(TNF-\selectlanguage{greek}α, \selectlanguage{english}IFN-\selectlanguage{greek}γ\selectlanguage{english}). Moreover, when given as an adjunct to conventional
chemotherapy, BEA enhanced bacterial clearance (36). Interestingly, this
synthetic hormone showed therapeutic benefit in patients with TB (2),
malaria (37) and in the Acquired Immune Deficiency Syndrome (AIDS). In
the HIV/AIDS trial BEA reduced the incidence of TB co-infection by
42\selectlanguage{ngerman}·2\% and the cumulative incidence of opportunistic infections (38).

In the present study a new water-miscible formulation of BEA was used.
Its ability to form a stable suspension in water is derived from a new
method of formulating the molecule. Water miscibility avoids the
problems associated with the organic solvents required to administer the
older, lipid soluble formulation. Mice treated with this new BEA
formulation showed similar results as the old synthetic analog,
producing significant increase of IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α, \selectlanguage{english}in coexistence with
pulmonary bacillary loads reduction, and now we demonstrated a new
activity, which is a significant pulmonary decrease in the expression of
11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and corticosterone. 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 which shifts the equilibrium to
inactive cortisone was overexpressed as well. Thus, BEA can induce CD4
Th1 cells and macrophages activation by direct activity, and as shown in
the present work, by the suppression of the local production of
corticosterone in the lungs.

Obesity is commonly associated with T2D and other manifestations of the
metabolic syndrome {[}1, 2{]}. The metabolic syndrome manifests as
obesity, hyperlipidemia and T2D, and is related to insulin resistance in
adipose tissue, skeletal muscle and liver {[}1, 3{]}. In obesity and
T2D, GCs are important pathogenic factors because they are antagonists
of insulin action. GC impair glucose uptake, enhance lipolysis and
hepatic gluconeogenesis and promote proteolysis. Moreover, GCs directly
inhibit pancreatic secretion of insulin, enhance glucose secretion by
inhibiting gluconeogenesis in the liver and oppose other metabolic
actions, such as insulin signaling and glucose uptake by inhibiting the
translocation of the glucose transporter GLUT4 to the plasma membrane
(19).

Our results show that in comparison with healthy control animals, mice
with T2D without Mtb infection exhibited higher 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 expression and
strong corticosterone immunostaining in bronchial epithelial cells and
in hepatocytes. BEA treatment decreased the expression of both enzyme
and hormone in lung and liver and corrected glucose concentration in
blood. Thus, BEA significantly improved metabolic abnormalities by
down-regulating 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 gene expression as one of the mechanisms. These
results are in agreement with previous publications, and contribute new
information such as the increase of corticosterone production in the
airways epithelium of T2D individuals which could be a factor for higher
predisposition of respiratory infections. One of these infections is TB.

Our results demonstrated a higher pulmonary bacillary load in T2D/TB
than in TB mice without T2D, as well as lower expression of IFN-\selectlanguage{greek}γ \selectlanguage{english}and
TNF-\selectlanguage{greek}α, \selectlanguage{english}in co-existence with higher expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 and strong
immunostaining to corticosterone in the pulmonary inflammatory
intra-alveolar infiltrate, particularly in foamy macrophages. Thus, it
seems that the higher pulmonary production of corticosterone mediated by
11-\selectlanguage{greek}β\selectlanguage{english}HSD1 is a factor that worsens the course of TB in T2D and this could
be a new therapeutic target.

In this regard, our results suggest that BEA can block 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 in the
lung and in the liver, becoming an effective treatment for this
co-morbidity. Trials for the use of BEA in the treatment of human TB,
T2D and the co-morbidity T2D/TB are now justified.

\textbf{Acknowledgments}

LT-MO is a PhD student from Programa de Doctorado en Ciencias
Biom\selectlanguage{ngerman}édicas, Universidad Nacional Autónoma de México (UNAM) and received
fellowship 433346 from CONACyT. This work was sponsored by Consejo
Nacional de Ciencia y Tecnología, Grant/Award Number: FC2015\selectlanguage{english}-1/115.

\textbf{Conclusion}

The study of T2D-TB comorbidity from the immunoendocrine point of view
is of utmost importance to understand the background of this binomial,
as well as to propose better strategies for the control of this
comorbidity. Active GCs represent a negative factor in the development
of both entities, with a negative effect on glucose metabolism and the
development of a non-protective immune response. Treatment with BEA
represents a novel strategy as an adjuvant in the treatment of TB alone
and comorbidity with T2D. For these reasons, it is of utmost importance
to continue with the study of the BEA through its escalation to clinical
studies to be used in patients.

\textbf{Conflict of interest}

We declare not conflict of interest during the course of this work.

\textbf{Author contributions}

LT-MO, AC and HP-R contributed to the background work and conceived the
experiments. LT-MO and RE-O performed, organized, and analyzed the
results. LT-MO, B-E, EB and MC-B experiments design and execution. BP-J
animal infections and drug administration. ME-D culture and preparation
of Mycobacterium tuberculosis H37Rv strain to infection and CFU
experiments, IT, NT, AT designed the hypercaloric diet. C-W, G-Y prepare
and supply BEA for the animal treatment.

\textbf{References}

1. Ayelign B, Negash M, Genetu M, Wondmagegn T, Shibabaw T.
Immunological Impacts of Diabetes on the Susceptibility of Mycobacterium
tuberculosis. \emph{J Immunol Res} (2019) \textbf{2019} :
doi:10.1155/2019/6196532

2. Ehlers S, Schaible UE. The granuloma in tuberculosis: Dynamics of a
host-pathogen collusion. \emph{Front Immunol} (2012) \textbf{3} :1--9.
doi:10.3389/fimmu.2012.00411

3. Meenakshi P, Ramya S, Lavanya J, Vijayalakshmi V, Sumanlatha G.
Effect of IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-12 and IL-10 cytokine production and mRNA expression
in tuberculosis patients with diabetes mellitus and their household
contacts. \emph{Cytokine} (2016) \textbf{81} :127--136.
doi:10.1016/j.cyto.2016.03.009

4. Cooper AM, Mayer-Barber KD, Sher A. Role of innate cytokines in
mycobacterial infection. \emph{Mucosal Immunol} (2011)\textbf{4}
:252--260. doi:10.1038/mi.2011.13

5. Boillat-Blanco N, Tumbo AMN, Perreau M, Amelio P, Ramaiya KL, Mganga
M, Schindler C, Gagneux S, Reither K, Probst-Hensch N, et al.
Hyperglycaemia is inversely correlated with live M. bovis BCG-specific
CD4+ T cell responses in Tanzanian adults with latent or active
tuberculosis. \emph{Immun Inflamm Dis} (2018) \textbf{6} :345--353.
doi:10.1002/iid3.222

6. Hodgson K, Morris J, Bridson T, Govan B, Rush C, Ketheesan N.
Immunological mechanisms contributing to the double burden of diabetes
and intracellular bacterial infections. \emph{Immunology}
(2015)\textbf{144} :171--185. doi:10.1111/imm.12394

7. Rey A del, Mahuad C V., Bozza V V., Bogue C, Farroni MA, Bay ML,
Bottasso OA, Besedovsky HO. Endocrine and cytokine responses in humans
with pulmonary tuberculosis. \emph{Brain Behav Immun} (2007)\textbf{21}
:171--179. doi:10.1016/j.bbi.2006.06.005

8. Fern\selectlanguage{ngerman}ández R, Díaz A, D'Attilio L, Bongiovanni B, Santucci N, Bertola
D, Besedovsky H, del Rey A, Bay ML, Bottasso O. An adverse
immune-endocrine profile in patients with tuberculosis and type 2
diabetes. \emph{Tuberculosis} (2016) \textbf{101} :95--101.
doi:10.1016/j.tube.2016.09.001

9. Talabér G, Jondal M, Okret S. Extra-adrenal glucocorticoid synthesis:
Immune regulation and aspects on local organ homeostasis. \emph{Mol Cell
Endocrinol} (2013) \textbf{380} :89--98. doi:10.1016/j.mce.2013.05.007

10. Loerz C, Maser E. The cortisol-activating enzyme 11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid
dehydrogenase type 1 in skeletal muscle in the pathogenesis of the
metabolic syndrome. \emph{J Steroid Biochem Mol Biol} (2017)\textbf{174}
:65--71. doi:10.1016/j.jsbmb.2017.07.030

11. Gomez-Sanchez EP, Ganjam V, Chen YJ, Liu Y, Clark SA, Gomez-Sanchez
CE. The 11\selectlanguage{greek}β \selectlanguage{english}hydroxysteroid dehydrogenase 2 exists as an inactive
dimer.\emph{Steroids} (2001) \textbf{66} :845--848.
doi:10.1016/S0039-128X(01)00119-2

12. Chapman KE, Coutinho AE, Gray M, Gilmour JS, Savill JS, Seckl JR.
The role and regulation of 11\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase type 1 in
the inflammatory response. \emph{Mol Cell Endocrinol} (2009)\textbf{301}
:123--131. doi:10.1016/j.mce.2008.09.031

13. Cidlowski DCJA. One Hormone , Two Actions : Anti- and
Pro-Inflammatory Effects of Glucocorticoids. (2015)\textbf{27709}
:20--32. doi:10.1159/000362724

14. Pandolfi J, Baz P, Fern\selectlanguage{ngerman}ández P, Discianni Lupi A, Payaslián F,
Billordo LA, Fainboim L, Arruvito L. Regulatory and effector T-cells are
differentially modulated by Dexamethasone. \emph{Clin Immunol}
(2013)\textbf{149} :400--410. doi:10.1016/j.clim.2013.09.008

15. Sapolsky SFS and RM. An Inflammatory Review of Glucocorticoid
Actions in the CNS. \emph{Bone} (2012) \textbf{23} :1--7.
doi:10.1016/j.bbi.2006.06.005

16. Masuzaki H, Paterson J, Shinyama H, Morton NM, Mullins JJ, Seckl JR,
Flier JS. A transgenic model of visceral obesity and the metabolic
syndrome. \emph{Science (80- )} (2001) \textbf{294} :2166--2170.
doi:10.1126/science.1066285

17. Apostolova G, Schweizer RAS, Balazs Z, Kostadinova RM, Odermatt A.
Dehydroepiandrosterone inhibits the amplification of glucocorticoid
action in adipose tissue. \emph{Am J Physiol - Endocrinol Metab}
(2005)\textbf{288} : doi:10.1152/ajpendo.00442.2004

18. Sävendahl L. The effect of acute and chronic stress on
growth.\emph{Sci Signal} (2012) \textbf{5} :9--11.
doi:10.1126/scisignal.2003484

19. Shukla R, Basu AK, Mandal B, Mukhopadhyay P, Maity A, Chakraborty S,
Devrabhai PK. 11\selectlanguage{greek}β \selectlanguage{english}Hydroxysteroid dehydrogenase - 1 activity in type 2
diabetes mellitus: A comparative study. \emph{BMC Endocr Disord}
(2019)\textbf{19} :1--9. doi:10.1186/s12902-019-0344-9

20. Chiodini I, Adda G, Scillitani A, Coletti F, Morelli V, Di Lembo S,
Epaminonda P, Masserini B, Beck-Peccoz P, Orsi E, et al. Cortisol
secretion in patients with type 2 diabetes: Relationship with chronic
complications. \emph{Diabetes Care} (2007) \textbf{30} :83--88.
doi:10.2337/dc06-1267

21. Buford TW, Willoughby DS. Impact of DHEA(S) and cortisol on immune
function in aging: A brief review. \emph{Appl Physiol Nutr Metab}
(2008)\textbf{33} :429--433. doi:10.1139/H08-013

22. Nicoletti F, Conrad D, Wang A, Pieters R, Mangano K, Van Heeckeren
A, White SK, Frincke J, Reading CL, Auci DL, et al.
16\selectlanguage{greek}α-\selectlanguage{english}Bromoepiandrosterone (HE2000) limits non-productive inflammation and
stimulates immunity in lungs. \emph{Clin Exp Immunol} (2009)\textbf{158}
:308--316. doi:10.1111/j.1365-2249.2009.04032.x

23. Ramos-Espinosa O, Islas-Weinstein L, Peralta-\selectlanguage{ngerman}Álvarez MP,
López-Torres MO, Hernández-Pando R. The use of immunotherapy for the
treatment of tuberculosis. \emph{Expert Rev Respir Med}
(2018)\textbf{12} :427--440. doi:10.1080/17476348.2018.1457439

24. Hernández-Pando R, Orozcoe H, Sampieri A, Pavón L, Velasquillo C,
Larriva-Sahd J, Alcocer JM, Madrid M V. Correlation between the kinetics
of Th1, Th2 cells and pathology in a murine model of experimental
pulmonary tuberculosis. \emph{Immunology} (1996) \textbf{89} :26--33.

25. Segura-Cerda CA, Marquina-Castillo B, Lozano-Ordaz V, Mata-Espinosa
D, Barrios-Payán JA, López-Torres MO, Aceves-Sánchez M de J,
Bielefeldt-Ohmann H, Hernández-Pando R, Flores-Valdez MA. BCG and
BCG\selectlanguage{greek}Δ\selectlanguage{english}BCG1419c protect type 2 diabetic mice against tuberculosis via
different participation of T and B lymphocytes, dendritic cells and
pro-inflammatory cytokines. \emph{npj Vaccines} (2020) \textbf{5}
:1--10. doi:10.1038/s41541-020-0169-6

26. Luo J, Quan J, Tsai J, Hobensack CK, Sullivan C, Hector R, Reaven
GM. Nongenetic mouse models of non-insulin-dependent diabetes
mellitus.\emph{Metabolism} (1998) \textbf{47} :663--668. doi:DOI:
10.1016/S0026-0495(98)90027-0

27. Rangel-Santiago JF, Baay-Guzman GJ, Duran-Padilla MA, Lopez-Bochm
KA, Garcia-Romero BL, Hernandez-Cueto DD, Pantoja-Escobar G, Vega MI,
Hernandez-Pando R, Huerta-Yepez S. A novel role of Yin-Yang-1 in
pulmonary tuberculosis through the regulation of the chemokine
CCL4.\emph{Tuberculosis} (2016) \textbf{96} :87--95.
doi:10.1016/j.tube.2015.10.013

28. Baay-Guzman GJ, Duran-Padilla MA, Rangel-Santiago J,
Tirado-Rodriguez B, Antonio-Andres G, Barrios-Payan J, Mata-Espinosa D,
Klunder-Klunder M, Vega MI, Hernandez-Pando R, et al. Dual role of
hypoxia-inducible factor 1 \selectlanguage{greek}α \selectlanguage{english}in experimental pulmonary tuberculosis: Its
implication as a new therapeutic target. \emph{Future Microbiol}
(2018)\textbf{13} :785--798. doi:10.2217/fmb-2017-0168

29. Zapata-Tarres M, Juarez-Villegas LE, Maldonado-Valenzuela A,
Baay-Guzman GJ, Lopez-Perez T V., Cabrera-Mu\selectlanguage{ngerman}ñoz L, Sadowinski-Pine S,
Huerta-Yepez S. Expression of YY1 in Wilms tumors with favorable
histology is a risk factor for adverse outcomes. \emph{Futur
Oncol}(2019) \textbf{15} :1231--1241. doi:10.2217/fon-2018-0764

30. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the
comparative CT method. \emph{Nat Protoc} (2008) \textbf{3} :1101--1108.
doi:10.1038/nprot.2008.73

31. Hernandez-Pando R, Orozco H, Aguilar D. Factors that deregulate the
protective immune response in tuberculosis. \emph{Arch Immunol Ther Exp
(Warsz)} (2009) \textbf{57} :355--367. doi:10.1007/s00005-009-0042-9

32. Bongiovanni B, Díaz A, D'Attilio L, Santucci N, Dídoli G, Lioi S,
Nannini LJ, Gardeñez W, Bogue C, Besedovsky H, et al. Changes in the
immune and endocrine responses of patients with pulmonary tuberculosis
undergoing specific treatment. \emph{Ann N Y Acad Sci}
(2012)\textbf{1262} :10--15. doi:10.1111/j.1749-6632.2012.06643.x

33. Hernandez-Pando R, Orozco H, Honour J, Silva P, Leyva R, Rook GAW.
Adrenal changes in murine pulmonary tuberculosis; a clue to
pathogenesis? \emph{FEMS Immunol Med Microbiol} (1995)\textbf{12}
:63--72. doi:10.1016/0928-8244(95)00051-8

34. Barrios-Payán J, Revuelta A, Mata-Espinosa D, Marquina-Castillo B,
Villanueva EB, Gutiérrez MEH, Pérez-Sánchez G, Pavón L, Hernandez-Pando
R. The contribution of the sympathetic nervous system to the
immunopathology of experimental pulmonary tuberculosis. \emph{J
Neuroimmunol} (2016) \textbf{298} :98--105.
doi:10.1016/j.jneuroim.2016.07.012

35. Irwin MR. Human psychoneuroimmunology: 20 Years of
discovery.\emph{Brain Behav Immun} (2008) \textbf{22} :129--139.
doi:10.1016/j.bbi.2007.07.013

36. Hernandez-Pando GAWR and R. Pathogenic Role, in Human and Murine
Tuberculosis, of Changes in the Pheripheral Metabolism of
Glucocorticoids ans Antiglucocorticoids. (1997) \textbf{22} :1--5.

37. Streber, M de la Luz, Orozco H, Arriaga K, Pavon L. The effects of
androstenediol and dehydroepiandrosterone on the course and cytokine
profile of tuberculosis in BALB/c mice. \emph{Immunology}(1998)234--241.

38. Leenstra T, ter~Kuile FO, Kariuki SK, Nixon CP, Oloo AJ, Kager PA,
Kurtis JD. Dehydroepiandrosterone Sulfate Levels Associated with
Decreased Malaria Parasite Density and Increased Hemoglobin
Concentration in Pubertal Girls from Western Kenya. \emph{J Infect
Dis}(2003) \textbf{188} :297--304. doi:10.1086/376508

39. Bozza V V., D'Attilio L, Mahuad C V., Giri AA, Del Rey A, Besedovsky
H, Bottasso O, Bay ML. Altered cortisol/DHEA ratio in tuberculosis
patients and its relationship with abnormalities in the
mycobacterial-driven cytokine production by peripheral blood mononuclear
cells. \emph{Scand J Immunol} (2007) \textbf{66} :97--103.
doi:10.1111/j.1365-3083.2007.01952.x

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.- Metabolic abnormalities and liver pathology of mice
with type 2 diabetes mellitus model vs non-diabetic animals
(control).}A) General description of the experimental groups. B) mice
were weighted weekly to monitor the weight gain induced by the
hypercaloric diet. Data are presented as a mean and standard derivation
of 5 mice per group for 16 weeks. C) Blood glucose levels were assessed
two and four weeks after administration of STZ intraperitoneally in
animals fed with a high-caloric diet treated with streptozotocin
(HFD-STZ) and in animals not treated with STZ and fed with Chow control
diet. Concentrations in sera of C) triglyceride, D) cholesterol, and E)
insulin in control and T2D groups, after four months of feeding with
hyper caloric diet and one month of STZ treatment. G) Effect of
hypoglycemic drugs on the control of serum glucose levels in T2D
animals, before and after 8 and 24 hours of oral administration of
metformin (Met) or glibenclamide (Gly), arrows indicate administration
times (0, 8 and 24 hours). Asterisk represents statistical significance
(one-way ANOVA, P \textless{}0.05, and two-way ANOVA for graph 1 B, 1 C,
and 1 G).H) Representative micrograph of liver tissue from a control
mouse (left figure) and extensive cytoplasmic vacuolization of
hepatocytes corresponding to steatosis in T2D mouse (right figure).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure. 2. Comparative course of pulmonary tuberculosis in
diabetic (T2D-TB) and non-diabetic (TB) mice.} A) Five mice per group
were sacrificed at the indicated time points (14, 21, 28, and 120
post-infection) and the right lungs were used to determine the bacterial
loads by counting colony-forming units (CFU). A significant increase in
the bacillary load of the T2D-TB group was observed after one and four
months of infection in comparison with the TB group. B) The comparative
survival rate of both groups. C) Comparative expression of TNF\selectlanguage{greek}α
\selectlanguage{english}determined by RT-PCR in the lungs of T2D/TB and TB groups, there is
significant lower expression after 120 days of infection in the former
group. Asterisk represent statistical significance p\textless{}0.05.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.- Gene expression and cellular sources of
glucocorticoids and their converting enzymes in late TB (day 120).} A)
Representative micrographs of immunohistochemical detection of
corticosterone in the epithelium of the lung airways and the alveolar
epithelium. Occasional bronchial epithelial cells show slight positivity
in the control mouse (CT), while in T2D mouse numerous bronchial cells
are strongly positive. Bronchial epithelium is negative in TB, and
T2D-TB; while in the pneumonic areas numerous macrophages show intense
positivity. B) Top panel show the comparative gene expression of
11-\selectlanguage{greek}β\selectlanguage{english}HSD1 determined by qPCR in the different experimental groups at day
120. T2D, TB, and T2D-TB groups show a significant increase in
comparison with the control group. Bottom panel show immunohistochemical
detection of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1, foamy vacuolated macrophages (inset) in pneumonic
areas from TB and T2D-TB show strong immunostaining. C) Representative
micrographs of immunohistochemistry detection of cortisone in bronchial
epithelial cells and alveoli. Alveolar epithelium show positive
immunostaining in the control mouse, while bronchial cells are negative
in all the conditions. TB mouse show strong immunostaining in alveolar
epithelium and in occasional inflammatory cells, while T2D and T2D/TB
are negative. D) Comparative gene expression of 11-\selectlanguage{greek}β\selectlanguage{english}HSD2 and its
representative immunohistochemistry detection. Asterisks represent
statistical significance (**** p\textless{} 0.0001, two- way ANOVA and
n.s. no significance).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.- Therapeutic effect of BEA in TB and T2D / TB
mice.}Groups of TB and T2D/TB mice after 60 days of intratracheal
infection with high dose Mtb strain H37Rv were treated during one or two
months with BEA, while the control group received only the vehicle.
Lungs were processed for the indicated determination. In comparison with
the control mice, in both TB and T2D/TB groups and particularly after 60
days of treatment, BEA induced significant decrease of pulmonary
bacillary loads and tissue damage (pneumonia), while after one month of
treatment BEA induced higher expression of the protective cytokines TNF\selectlanguage{greek}α
\selectlanguage{english}and IFN-\selectlanguage{greek}γ. \selectlanguage{english}In the spleen, treatment with BEA did not produce significant
changes in the weight and CFUs in TB mice, while in T2D/TB mice induced
after two months of treatment significant decrease of bacillary burdens
and higher weight increase. Asterisks represent statistical significance
(**** p\textless{} 0.0001, two- way ANOVA and n.s. no significance).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.- Effect of BEA on lung glucocorticoids production and
the gene expression of their converting enzymes in TB and T2D-TB
mice.}A) Representative low power and mild power micrographs of
immunohistochemistry detection of corticosterone, in both groups after
one and two months of BEA treatment. B) In comparison with the control
group, the digital pathology analysis showed significant lower
immunostaining of corticosterone induced by BEA treatment after one and
two months in TB mice and T2D-TB mice. C) BEA treatment induced lower
11b-HSD1 gene expression in TB and T2D-TB in comparison with
vehicle-treated animals. D) Representative micrographs of cortisone
detection by immunohistochemistry in the lungs of TB and T2D mice
treated with BEA at one and two months of infection. E) The digital
pathology study shows higher expression of cortisone after one month of
treatment in the TB and T2D/TB groups. F) Gene expression of 11b-HSD2
corticosterone converting enzyme after BEA treatment in TB and T2D-TB
animals. Asterisk represents statistical significance
(p\textless{}0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6.- Effect of BEA in the liver of T2D and T2D-TB
mice.}Liver samples from groups of five T2D and T2D-TB mice treated with
BEA for one month and their respective control mice that received only
the vehicle, were used to isolate total RNA and determine the expression
of 11-\selectlanguage{greek}β\selectlanguage{english}HSD1 (A) and 11 -\selectlanguage{greek}β\selectlanguage{english}HSD2 (B) by RT-PCR. C) Treatment with BEA
corrected hyperglycemia in T2D and T2D-TB mice. Asterisks represent
statistical significance (*** p \textless{}0.0001, **** p
\textless{}0.0001, two-way ANOVA, n.s. without significance). D) Liver
tissue sections stained with H / E show steatosis in the control mouse,
which was eliminated by treatment with BEA in T2D and T2D-TB mice.

\selectlanguage{english}
\FloatBarrier
\end{document}

